Method of determining a chemotherapeutic regimen based on glutathione-s-transferase pi

ABSTRACT

The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing GST-pi expression levels in fixed or fixed and paraffin embedded tissues and prognosticate the probable resistance or sensitivity of a patient&#39;s tumor to treatment with platinum-based therapies by examination of the amount of GST-pi mRNA in a patient&#39;s tumor cells and comparing it to a predetermined threshold expression level. More specifically, the invention provides to oligonucleotide primer pair GST-piand methods comprising their use for detecting levels of GST-pi mRNA.

FIELD OF THE INVENTION

[0001] The present invention relates to prognostic methods which areuseful in medicine, particularly cancer chemotherapy. More particularly,the invention relates to assessment of tumor cell gene expression in apatient. The resistance of tumor cells to chemotherapeutic agents thattarget DNA, especially agents that damage DNA in the manner ofplatinating agents is assayed by examining the mRNA expressed from genesinvolved in DNA repair in humans.

BACKGROUND OF THE INVENTION

[0002] Cancer arises when a normal cell undergoes neoplastictransformation and becomes a malignant cell. Transformed (malignant)cells escape normal physiologic controls specifying cell phenotype andrestraining cell proliferation. Transformed cells in an individual'sbody thus proliferate, forming a tumor. When a tumor is found, theclinical objective is to destroy malignant cells selectively whilemitigating any harm caused to normal cells in the individual undergoingtreatment.

[0003] Chemotherapy is based on the use of drugs that are selectivelytoxic (cytotoxic) to cancer cells. Several general classes ofchemotherapeutic drugs have been developed, including drugs thatinterfere with nucleic acid synthesis, protein synthesis, and othervital metabolic processes. These generally are referred to asantimetabolite drugs. Other classes of chemotherapeutic drugs inflictdamage on cellular DNA. Drugs of these classes generally are referred toas genotoxic. Susceptibility of an individual neoplasm to a desiredchemotherapeutic drug or combination of drugs often, however, can beaccurately assessed only after a trial period of treatment. The timeinvested in an unsuccessful trial period poses a significant risk in theclinical management of aggressive malignancies.

[0004] The repair of damage to cellular DNA is an important biologicalprocess carried out by a cell's enzymatic DNA repair machinery.Unrepaired lesions in a cell's genome can impede DNA replication, impairthe replication fidelity of newly synthesized DNA and/or hinder theexpression of genes needed for cell survival. Thus, genotoxic drugsgenerally are considered more toxic to actively dividing cells thatengage in DNA synthesis than to quiescent, nondividing cells. Normalcells of many body tissues are quiescent and commit infrequently tore-enter the cell cycle and divide. Greater time between rounds of celldivision generally is afforded for the repair of DNA damage in normalcells inflicted by chemotherapeutic genotoxins. As a result, someselectivity is achieved for the killing of cancer cells. Many treatmentregimens reflect attempts to improve selectivity for cancer cells bycoadministering chemotherapeutic drugs belonging to two or more of thesegeneral classes.

[0005] Because effective chemotherapy in solid tumors usually requires acombination of agents, the identification and quantification ofdeterminants of resistance or sensitivity to each single drug has becomean important tool to design individual combination chemotherapy.

[0006] Two widely used genotoxic anticancer drugs that have been shownto damage cellular DNA are cisplatin (DDP) and carboplatin. Cisplatinand/or carboplatin currently are used in the treatment of selected,diverse neoplasms of epithelial and mesenchymal origin, includingcarcinomas and sarcomas of the respiratory, gastrointestinal andreproductive tracts, of the central nervous system, and of squamousorigin in the head and neck. Cisplatin in combination with other agentsis currently preferred for the management of testicular carcinoma, andin many instances produces a lasting remission. (Loehrer et al.,1984,100Ann. Int. Med. 704). Cisplatin (DDP) disrupts DNA structure throughformation of intrastrand adducts. Resistance to platinum agents such asDDP has been attributed to enhanced tolerance to platinum adducts,decreased drug accumulation, or enhanced DNA repair. Although resistanceto DDP is multifactoral, alterations in DNA repair mechanisms probablyplay a significant role.

[0007] The glutathione-S-transferase (GST) family of proteins isinvolved in detoxification of cytotoxic drugs. By catalyzing theconjugation of toxic and carcinogenic electrophilic molecules withglutathione the GST enzymes protect cellular macromolecules from damage(Boyer et al., Preparation, characterization and properties ofglutathione S-transferases. In: Zakim D, Vessey D (eds.) BiochemicalPharmacology and Toxicology. New York, N.Y.: John Wiley and Sons,1985.). A certain isomeric type of these proteins, the glutathioneS-transferase Pi (GST-pi, also to be interchangeably refered to as GSTP1or GST-π herein) is widely expressed in human epithelial tissues and hasbeen demonstrated to be over-expressed in several tumors (Terrier etal., Am J Pathol 1990; 137: 845-853; Moscow et al., Cancer Res 1989; 49:1422-1428). Increased GST-pi levels have been found in drug resistanttumors, although the exact mechanism remains unclear (Tsuchida et al.,Crit Rev Biochem Mol Biol 1992; 27: 337-384). Previous studies havesuggested that low expression of GST protein (not mRNA) is associatedwith response to platinum-based chemotherapy (Nishimura et al., Cancer.Clin Cancer Res 1996; 2:1859-1865; Tominaga, et al., Am. J. Gastro.94:1664-1668, 1999; Kase, et al., Acta Cytologia. 42: 1397-1402, 1998).However, these studies did not measure quantitative gene expression, butused a semi-quantitative immunohistochemical staining method to measureprotein levels. However, quantitative GST-pi gene expressionmeasurements are needed to achieve a very effective prognostication.

[0008] Most pathological samples are routinely fixed andparaffin-embedded (FPE) to allow for histological analysis andsubsequent archival storage. Thus, most biopsy tissue samples are notuseful for analysis of gene expression because such studies require ahigh integrity of RNA so that an accurate measure of gene expression canbe made. Currently, gene expression levels can be only qualitativelymonitored in such fixed and embedded samples by usingimmunohistochemical staining to monitor protein expression levels.

[0009] Until now, quantitative gene expression studies including thoseof GST-pi expression have been limited to reverse transcriptasepolymerase chain reaction (RT-PCR) amplification of RNA from fresh orfrozen tissue.

[0010] The use of frozen tissue by health care professionals posessubstantial inconveniences. Rapid biopsy delivery to avoid tissue andsubsequent mRNA degradation is the primary concern when planning anyRNA-based quantitative genetic marker assay. The health careprofessional performing the biopsy, must hastily deliver the tissuesample to a facility equipped to perform an RNA extraction protocolimmediately upon tissue sample receipt. If no such facility isavailable, the clinician must promptly freeze the sample in order toprevent mRNA degradation. In order for the diagnostic facility toperform a useful RNA extraction protocol prior to tissue and RNAdegradation, the tissue sample must remain frozen until it reaches thediagnostic facility, however far away that may be. Maintenance of frozentissue integrity during transport using specialized couriers equippedwith liquid nitrogen and dry ice, comes only at a great expense.

[0011] Routine biopsies generally comprise a heterogenous mix of stromaland tumorous tissue. Unlike with fresh or frozen tissue, FPE biopsytissue samples are readily microdissected and separated into stromal andtumor tissue and therefore, offer andvantage over the use of fresh orfrozen tissue. However, isolation of RNA from fixed tissue, andespecially fixed and paraffin embedded tissue, results in highlydegraded RNA, which is generally not applicable to gene expressionstudies.

[0012] A number of techniques exist for the purification of RNA frombiological samples, but none is reliable for isolation of RNA from FPEsamples. For example, Chomczynski (U.S. Pat. No. 5,346,994) describes amethod for purifying RNA from tissues based on a liquid phase separationusing phenol and guanidine isothiocyanate. A biological sample ishomogenized in an aqueous solution of phenol and guanidineisothiocyanate and the homogenate thereafter mixed with chloroform.Following centrifugation, the homogenate separates into an organicphase, an interphase and an aqueous phase. Proteins are sequestered inthe organic phase, DNA in the interphase, and RNA in the aqueous phase.RNA can be precipitated from the aqueous phase. Unfortunately, thismethod is not applicable to fixed and paraffin-embedded (FPE) tissuesamples.

[0013] Other known techniques for isolating RNA typically utilize eitherguanidine salts or phenol extraction, as described for example inSambrook, J. et al., (1989) at pp. 7.3-7.24, and in Ausubel, F. M. etal., (1994) at pp. 4.0.3-4.4.7. Again, none of the known methodsprovides reproducible quantitative results in the isolation of RNA fromparaffin-embedded tissue samples.

[0014] Techniques for the isolation of RNA from paraffin-embeddedtissues are thus particularly needed for the study of gene expression intumor tissues, since expression levels of certain receptors or enzymescan be used to determine the likelihood of success of a particulartreatment.

[0015] There is a need for a method of quantifying GST-pi mRNA fromparaffinized tissue in order to provide an early prognosis for proposedgenotoxic cancer therapies. As a result, there has been a concerted yetunsuccessful effort in the art to obtain a quantification of GST-piexpression in fixed and paraffinized (FPE) tissue. Accordingly, it isthe object of the invention to provide a method for assessing GST-pilevels in tissues fixed and paraffin-embedded (FPE) and prognosticatethe probable resistance of a patient's tumor to treatment with DNAdamaging agents, creating the type of lesions in DNA that are created byDNA platinating agents, by examination of the amount of GST-pi mRNA in apatient's tumor cells and comparing it to a predetermined thresholdexpression level.

SUMMARY OF THE INVENTION

[0016] In one aspect of the invention there is provided a method forassessing levels of expression of GST-pi mRNA obtained from fixed andparaffin-embedded (FPE) fixed and paraffin-embedded (FPE) tumor cells.

[0017] In another aspect of the invention there is provided a method ofquantifying the amount of GST-pi mRNA expression relative to an internalcontrol from a fixed and paraffin-embedded (FPE) tissue sample. Thismethod includes isolation of total mRNA from said sample and determiningthe quantity of GST-pi mRNA relative to the quantity of an internalcontrol gene's mRNA.

[0018] In an embodiment of this aspect of the invention, there areprovided oligonucleotide primers having the sequence of GST-F (SEQ IDNO: 1) or GST-R (SEQ ID NO:2) and sequences substantially identicalthereto. The invention also provides for oligonucleotide primers havinga sequence that hybridizes to SEQ ID NO: 1 or SEQ ID NO:2 or theircomplements under stringent conditions.

[0019] In yet another aspect of the invention there is provided a methodfor determining a chemotherapeutic regimen for a patient, comprisingisolating RNA from a fixed and paraffin-embedded (FPE) tumor sample;determining a gene expression level of GST-pi in the sample; comparingthe GST-pi gene expression levels in the sample with a predeteriminedthreshold level for the GST-pi gene; and determining a chemotherapeuticregimen based on results of the comparison of the GST-pi gene expressionlevel with the predetermined threshold level.

[0020] The invention further relates to a method of normalizing theuncorrected gene expression (UGE) of GST-pi relative to an internalcontrol gene in a tissue sample analyzed using TaqMan® technology toknown GST-pi expression levels relative to an internal control fromsamples analyzed by pre-TaqMan® technology.

DESCRIPTION OF THE DRAWING

[0021]FIG. 1 shows and association between survival and GST-pi correctedrelative mRNA expression in patients with esophagocardiac adenocarcinomatreated with 5-FU and cisplatin. Patients with GST-pi values above themedian/threshold value had a survival advantage compared to those withpatients with values below the median/threshold. Censored values aredenoted by a tick.

[0022]FIG. 2 is a graph showing survival analysis confined to patientswith TNM Stage II esophagocardiac adenocarcinoma

[0023]FIG. 3 is a graph showing survival analysis confined to patientswith Stage IV esophagocardiac adenocarcinoma

[0024]FIG. 4 is a chart illustrating how to calculate GST-pi expressionrelative to an internal control gene. The chart contains data obtainedwith two test samples, (unknowns 1 and 2), and illustrates how todetermine the uncorrected gene expression data (UGE). The chart alsoillustrates how to normalize UGE generated by the TaqMan® instrumentwith known relative GST-pi values determined by pre-TaqMan® technology.This is accomplished by multiplying UGE to a correction factorK_(GST-pi). The internal control gene in the figure is β-actin and thecalibrator RNA is Human Liver Total RNA (Stratagene, Cat. #735017).

[0025]FIG. 5 shows the oligonucleotide primers used in the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention resides in part in the finding that theamount of GST-pi mRNA is correlated with increased sensitivity to DNAplatinating agents. Tumors expressing high levels of GST-pi mRNA areconsidered likely to be sensitive to platinum-based chemotherapy.Conversely, those tumors expressing low amounts of GST-pi mRNA arelikely to be insensitive to platinum-based chemotherapy. A patient'srelative expression of tumor GST-pi mRNA is judged by comparing it to apredetermined threshold expression level. Such sensitivity or lackthereof to DNA platinating agents is determined by a patient'ssurvivability.

[0027] The invention relates to a method of quantifying the amount ofGST-pi mRNA expression in fixed and paraffin-embedded (FPE) tissuerelative to gene expression of an internal control. The presentinventors have developed oligonucleotide primers that allow accurateassessment of GST-pi expression in tissues that have been fixed andembedded. The invention oligonucleotide primers, GST-F (SEQ ID NO: 1),GST-R (SEQ ID NO: 2), or oligonucleotide primers substantially identicalthereto, preferably are used together with RNA extracted from fixed andparaffin embedded (FPE) tumor samples. This measurement of GST-pi geneexpression may then be used for prognosis of platinum-basedchemotherapy.

[0028] This embodiment of the invention involves first, a method forreliable extraction of RNA from an FPE sample and second, determinationof the content of GST-pi mRNA in the sample by using a pair ofoligonucleotide primers, preferably oligionucleotide primer pair GST-F(SEQ ID NO: 1) and GST-R (SEQ ID NO: 2), or oligonucleotidessubstantially identical thereto, for carrying out reverse transcriptasepolymerase chain reaction. RNA is extracted from the FPE cells by any ofthe methods for mRNA isolation from such samples as described in U.S.patent application Ser. No. 09/469,338, filed Dec. 20, 1999, and ishereby incorporated by reference in its entirety.

[0029] The present method can be applied to any type of tissue from apatient. For examination of sensitivity of tumor tissue, it ispreferable to examine the tumor tissue. In a preferred embodiment, aportion of normal tissue from the patient from which the tumor isobtained, is also examined.

[0030] The methods of the present invention can be applied over a widerange of tumor types. This allows for the preparation of individual“tumor expression profiles” whereby expression levels of GST-pi aredetermined in individual patient samples and response to variouschemotherapeutics is predicted. Preferably, the methods of the inventionare applied to solid tumors, most preferably esophogocardiac tumors. Forapplication of some embodiments of the invention to particular tumortypes, it is preferable to confirm the relationship of GST-pi geneexpression levels to clinical resistance by compiling a data-set thatenables correlation of a particular GST-pi expression and clinicalresistance to platinum-based chemotherapy.

[0031] A “predetermined threshold level”, as defined herein, is a levelof GST-pi expression above which it has been found that tumors arelikely to be sensitive to a platinum-based chemotherapeutic regimen.Expression levels below this threshold level are likely to be found intumors insensitive to platinum-based chemotherapeutic regimen. The rangeof corrected relative expression of GST-pi, expressed as a ratio ofGST-pi:β-actin, among tumors responding to a platinum-basedchemotherapeutic regimen is more than about 1.0×10⁻³. Tumors that do notrespond to a platinum-based chemotherapeutic regimen have relativeexpression of GST-pi: β-actin ratio below about 1.0×10⁻³. FIG. 1.However, the present invention is not limited to the use of β-actin asan internal control gene.

[0032] In performing the method of this embodiment of the presentinvention, tumor cells are preferably isolated from the patient. Solidor lymphoid tumors or portions thereof are surgically resected from thepatient or obtained by routine biopsy. RNA isolated from frozen or freshsamples is extracted from the cells by any of the methods typical in theart, for example, Sambrook, Fischer and Maniatis, Molecular Cloning, alaboratory manual, (2nd ed.), Cold Spring Harbor Laboratory Press, NewYork, (1989). Preferably, care is taken to avoid degradation of the RNAduring the extraction process.

[0033] However, tissue obtained from the patient after biopsy is oftenfixed, usually by formalin (formaldehyde) or gluteraldehyde, forexample, or by alcohol immersion. Fixed biological samples are oftendehydrated and embedded in paraffin or other solid supports known tothose of skill in the art. Non-embedded, fixed tissue may also be usedin the present methods. Such solid supports are envisioned to beremovable with organic solvents for example, allowing for subsequentrehydration of preserved tissue.

[0034] RNA is extracted from the FPE cells by any of the methods asdescribed in U.S. patent application Ser. No. 09/469,338, filed Dec. 20,1999, which is hereby incorporated by reference in its entirety. Fixedand paraffm-embedded (FPE) tissue samples as described herein refers tostorable or archival tissue samples. RNA may be isolated from anarchival pathological sample or biopsy sample which is firstdeparaffinized. An exemplary deparaffinization method involves washingthe paraffinized sample with an organic solvent, such as xylene, forexample. Deparaffinized samples can be rehydrated with an aqueoussolution of a lower alcohol. Suitable lower alcohols, for exampleinclude, methanol, ethanol, propanols, and butanols. Deparaffinizedsamples may be rehydrated with successive washes with lower alcoholicsolutions of decreasing concentration, for example. Alternatively, thesample is simultaneously deparaffinized and rehydrated. RNA is thenextracted from the sample.

[0035] For RNA extraction, the fixed or fixed and deparaffinized samplescan be homogenized using mechanical, sonic or other means ofhomogenization. Rehydrated samples may be homogenized in a solutioncomprising a chaotropic agent, such as guanidinium thiocyanate (alsosold as guanidinium isothiocyanate). Homogenized samples are heated to atemperature in the range of about 50 to about 100° C. in a chaotropicsolution, which contains an effective amount of a chaotropic agent, suchas a guanidinium compound. A preferred chaotropic agent is guanidiniumthiocyanate.

[0036] An “effective concentration of chaotropic agent” is chosen suchthat at an RNA is purified from a paraffin-embedded sample in an amountof greater than about 10-fold that isolated in the absence of achaotropic agent. Chaotropic agents include: guanidinium compounds,urea, formamide, potassium iodiode, potassium thiocyantate and similarcompounds. The preferred chaotropic agent for the methods of theinvention is a guanidinium compound, such as guanidinium isothiocyanate(also sold as guanidinium thiocyanate) and guanidinium hydrochloride.Many anionic counterions are useful, and one of skill in the art canprepare many guanidinium salts with such appropriate anions. Theeffective concentration of guanidinium solution used in the inventiongenerally has a concentration in the range of about 1 to about 5M with apreferred value of about 4M. If RNA is already in solution, theguanidinium solution may be of higher concentration such that the finalconcentration achieved in the sample is in the range of about 1 to about5M. The guanidinium solution also is preferably buffered to a pH ofabout 3 to about 6, more preferably about 4, with a suitable biochemicalbuffer such as Tris-Ci. The chaotropic solution may also containreducing agents, such as dithiothreitol (DTT) and β-mercaptoethanol(BME). The chaotropic solution may also contain RNAse inhibitors.

[0037] Homogenized samples may be heated to a temperature in the rangeof about 50 to about 100° C. in a chaotropic solution, which contains aneffective amount of a chaotropic agent, such as a guanidinium compound.A preferred chaotropic agent is guanidinium thiocyanate.

[0038] RNA is then recovered from the solution by, for example, phenolchloroform extraction, ion exchange chromatography or size-exclusionchromatography. RNA may then be further purified using the techniques ofextraction, electrophoresis, chromatography, precipitation or othersuitable techniques.

[0039] The quantification of GST-pi mRNA from purified total mRNA fromfresh, frozen or fixed is preferably carried out usingreverse-transcriptase polymerase chain reaction (RT-PCR) methods commonin the art, for example. Other methods of quantifying of GST-pi mRNAinclude for example, the use of molecular beacons and other labeledprobes useful in multiplex PCR. Additionally, the present inventionenvisages the quantification of GST-pi mRNA via use of PCR-free systemsemploying, for example fluorescent labeled probes similar to those ofthe Invader® Assay (Third Wave Technologies, Inc.). Most preferably,quantification of GST-pi cDNA and an internal control or house keepinggene (e.g. β-actin) is done using a fluorescence based real-timedetection method (ABI PRISM 7700 or 7900 Sequence Detection System[TaqMan®], Applied Biosystems, Foster City, Calif.) or similar system asdescribed by Heid et al., (Genome Res 1996;6:986-994) and Gibson etal.(Genome Res 1996;6:995-1001). The output of the ABI 7700 (TaqMan®Instrument) is expressed in Ct's or “cycle thresholds”. With the TaqMan®system, a highly expressed gene having a higher number of targetmolecules in a sample generates a signal with fewer PCR cycles (lowerCt) than a gene of lower relative expression with fewer target molecules(higher Ct).

[0040] As used herein, a “house keeping” gene or “internal control” ismeant to include any constitutively or globally expressed gene whosepresence enables an assessment of GST-pi mRNA levels. Such an assessmentcomprises a determination of the overall constitutive level of genetranscription and a control for variations in RNA recovery.“House-keeping” genes or “internal controls” can include, but are notlimited to the cyclophilin gene, β-actin gene, the transferrin receptorgene, GAPDH gene, and the like. Most preferably, the internal controlgene is β-actin gene as described by Eads et al., Cancer Research 1999;59:2302-2306.

[0041] A control for variations in RNA recovery requires the use of“calibrator RNA.” The “calibrator RNA” is intended to be any availablesource of accurately pre-quantified control RNA. Preferably, AdultColon, Disease Human Total RNA, (Cat. No. #735263) from Stratagene, isused.

[0042] “Uncorrected Gene Expression (UGE)” as used herein refers to thenumeric output of GST-pi expression relative to an internal control genegenerated by the TaqMan® instrument. The equation used to determine UGEis shown in Example 3, and illustrated with sample calculations in FIG.4.

[0043] A further aspect of this invention provides a method to normalizeuncorrected gene expression (UGE) values acquired from the TaqMan®instrument with “known relative gene expression” values derived fromnon-TaqMan® technology. Preferably, the known non-TaqMan® derivedrelative GST-pi: β-actin expression values are normalized with TaqMan®derived GST-pi UGE values from a tissue sample.

[0044] “Corrected Relative GST-pi Expression” as used herein refers tonormalized GST-pi expression whereby UGE is multiplied with a GST-pispecific correction factor (K_(GST-pi)), resulting in a value that canbe compared to a known range of GST-pi expression levels relative to aninternal control gene. Example 3 and FIG. 4 illustrate thesecalculations in detail. These numerical values allow the determinationof whether or not the “Corrected Relative GST-pi Expression” of aparticular sample falls above or below the “predetermined threshold”level. The predetermined threshold level of Corrected Relative GST-piExpression to β-actin level is about 1.0×10⁻³. K_(GST-pi) specific forGST-pi, the internal control β-actin and calibrator Adult Colon, DiseaseHuman Total RNA, (Cat. No. #735263) from Stratagene, is 7.28×10⁻³.

[0045] “Known relative gene expression” values are derived frompreviously analyzed tissue samples and are based on the ratio of theRT-PCR signal of a target gene to a constitutively expressed internalcontrol gene (e.g. β-Actin, GAPDH, etc.). Preferably such tissue samplesare formalin fixed and paraffin-embedded (FPE) samples and RNA isextracted from them according to the protocol described in Example 1 andin U.S. patent application Ser. No. 09/469,338, filed Dec. 20, 1999,which is hereby incorporated by reference in its entirety. To quantifygene expression relative to an internal control standard quantitativeRT-PCR technology known in the art is used. Pre-TaqMan®) technology PCRreactions are run for a fixed number of cycles (i.e., 30) and endpointvalues are reported for each sample. These values are then reported as aratio of GST-pi expression to β-actin expression. See U.S. Pat. No.5,705,336 to Reed et al.

[0046] K_(GST-Pi) may be determined for an internal control gene otherthan β-actin and/or a calibrator RNA different than Adult Colon, DiseaseHuman Total RNA, (Cat. No. #735263) from Stratagene. To do so, one mustcalibrate both the internal control gene and the calibrator RNA totissue samples for which GST-pi expression levels relative to thatparticular internal control gene have already been determined (i.e.,“known relative gene expression”). Preferably such tissue samples areformalin fixed and paraffin-embedded (FPE) samples and RNA is extractedfrom them according to the protocol described in Example 1 and in U.S.patent application Ser. No. 09/469,338, filed Dec. 20, 1999, which ishereby incorporated by reference in its entirety. Such a determinationcan be made using standard pre-TaqMan®, quantitative RT-PCR techniqueswell known in the art. Upon such a determination, such samples have“known relative gene expression” levels of GST-pi useful in thedetermining a new K_(GST-Pi) specific for the new internal controland/or calibrator RNA as described in Example 3.

[0047] The methods of the invention are applicable to a wide range oftissue and tumor types and so can be used for assessment of clinicaltreatment of a patient and as a diagnostic or prognostic tool for arange of cancers including breast, head and neck, lung, esophageal,colorectal, and others. In a preferred embodiment, the present methodsare applied to prognosis of esophagocardiac adenocarcinoma.

[0048] Pre-chemotherapy treatment tumor biopsies are usually availableonly as fixed paraffin embedded (FPE) tissues, generally containing onlya very small amount of heterogeneous tissue. Such FPE samples arereadily amenable to microdissection, so that GST-pi gene expression maybe determined in tumor tissue uncontaminated with stromal tissue.Additionally, comparisons can be made between stromal and tumor tissuewithin a biopsy tissue sample, since such samples often contain bothtypes of tissues.

[0049] Generally, any oligonucleotide pair that flanks a region ofGST-pi gene may be used to carry out the methods of the invention.Primers hybridizing under stringent conditions to a region of the GST-pigene for use in the present invention will amplify a product between20-1000 base pairs, preferably 50-100 base pairs, most preferably lessthan 100 base pairs.

[0050] The invention provides specific oligonucleotide primers pairs andoligonucleotide primers substantially identical thereto, that allowparticularly accurate assessment of GST-pi expression in FPE tissues.Preferable are oligonucleotide primers, GST-F (SEQ ID NO: 1) and GST-R(SEQ ID NO: 2), (also referred to herein as the oligonucleotide primerpair GST) and oligonucleotide primers substantially identical thereto.The oliogonucleotide primers GST-F (SEQ ID NO: 1) and GST-R, (SEQ ID NO:2) have been shown to be particularly effective for measuring GST-pimRNA levels using RNA extracted from the FPE cells by any of the methodsfor mRNA isolation, for example as described Example 1 and in U.S.patent application Ser. No. 09/469,338, filed Dec. 20, 1999, which ishereby incorporated by reference in its entirety.

[0051] “Substantially identical” in the nucleic acid context as usedherein, means hybridization to a target under stringent conditions, andalso that the nucleic acid segments, or their complementary strands,when compared, are the same when properly aligned, with the appropriatenucleotide insertions and deletions, in at least about 60% of thenucleotides, typically, at least about 70%, more typically, at leastabout 80%, usually, at least about 90%, and more usually, at least,about 95-98% of the nucleotides. Selective hybridization exists when thehybridization is more selective than total lack of specificity. See,Kanehisa, Nucleic Acids Res., 12:203-213 (1984).

[0052] This invention includes substantially identical oligonucleotidesthat hybridize under stringent conditions (as defined herein) to all ora portion of the oligonucleotide primer sequence of GST-F (SEQ ID NO:1), its complement or GST-R (SEQ ID NO: 2), or its complement.

[0053] Under stringent hybridization conditions, only highlycomplementary, i.e., substantially similar nucleic acid sequenceshybridize. Preferably, such conditions prevent hybridization of nucleicacids having.4 or more mismatches out of 20 contiguous nucleotides, morepreferably 2 or more mismatches out of 20 contiguous nucleotides, mostpreferably one or more mismatch out of 20 contiguous nucleotides.

[0054] The hybridizing portion of the nucleic acids is typically atleast 10 (e.g., 15) nucleotides in length. The hybridizing portion ofthe hybridizing nucleic acid is at least about 80%, preferably at leastabout 95%, or most preferably about at least 98%, identical to thesequence of a portion or all of oligonucleotide primer GST-F (SEQ ID NO:1), its complement or GST-R (SEQ ID NO: 2), or its complement.

[0055] Hybridization of the oligonucleotide primer to a nucleic acidsample under stringent conditions is defined below. Nucleic acid duplexor hybrid stability is expressed as a melting temperature (T_(m)), whichis the temperature at which the probe dissociates from the target DNA.This melting temperature is used to define the required stringencyconditions. If sequences are to be identified that are substantiallyidentical to the probe, rather than identical, then it is useful tofirst establish the lowest temperature at which only homologoushybridization occurs with a particular concentration of salt (e.g. SSCor SSPE). Then assuming that 1% mismatching results in a 1° C. decreasein T_(m), the temperature of the final wash in the hybridizationreaction is reduced accordingly (for example, if sequences having >95%identity with the probe are sought, the final wash temperature isdecrease by 5° C.). In practice, the change in T_(m) can be between 0.5°C. and 1.5° C. per 1% mismatch.

[0056] Stringent conditions involve hybridizing at 68° C. in5×SSC/5×Denhart's solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS atroom temperature. Moderately stringent conditions include washing in3×SSC at 42° C. The parameters of salt concentration and temperature bevaried to achieve optimal level of identity between the primer and thetarget nucleic acid. Additional guidance regarding such conditions isreadily available in the art, for example, Sambrook, Fischer andManiatis, Molecular Cloning, a laboratory manual, (2nd ed.), Cold SpringHarbor Laboratory Press, New York, (1989) and F. M. Ausubel et al eds.,Current Protocols in Molecular Biology, John Wiley and Sons (1994).

[0057] Oligonucleotide primers disclosed herein are capable of allowingaccurate assessment of GST-pi gene expression in a fixed or fixed andparaffin embedded tissue, as well as frozen or fresh tissue. This isdespite the fact that RNA derived from FPE samples is more fragmentedrelative to that of fresh or frozen tissue. Thus, the methods of theinvention are suitable for use in assaying GST-pi expression levels inFPE tissue where previously there existed no way to assay GST-pi geneexpression using fixed tissues.

[0058] From the measurement of the amount of GST-pi mRNA that isexpressed in the tumor, the skilled practitioner can make a prognosisconcerning clinical resistance of a tumor to a particular genotoxin,preferably a platinum-based chemotherapy, or to a chemotherapy inducinga similar type of DNA damage. Platinum-based chemotherapies cause a“bulky adduct” of the DNA, wherein the primary effect is to distort thethree-dimensional conformation of the double helix. Such compounds aremeant to be administered alone, or together with other chemotherapiessuch as gemcitabine (Gem) or 5-Fluorouracil (5-FU).

[0059] Many compounds are commonly given with platinum-basedchemotherapy agents. For example, BEP (bleomycin, etoposide, cisplatin)is used for testicular cancer, MVAC (methotrexate, vinblastine,doxorubicin, cisplatin) is used for bladder cancer, MVP (mitomycin C,vinblastine, cisplatin) is used for non-small cell lung cancertreatment. Many studies have documented interactions betweenplatinum-containing agents. Therapeutic drug synergism, for example, hasbeen reported for many drugs potentially included in a platinum basedchemotherapy. A very short list of recent references for this includethe following: Okamoto et al., Urology 2001; 57:188-192.; Tanaka et al.,Anticancer Research 2001; 21:313-315; Slamon et al., Seminars inOncology 2001; 28:13-19; Lidor et al., Journal of Clinical Investigation1993; 92:2440-2447; Leopold et al., NCI Monographs 1987;99-104; Ohta etal., Cancer Letters 2001; 162:39-48; van Moorsel et al., British Journalof Cancer 1999; 80:981-990.

[0060] Platinum-based genotoxic chemotherapies comprises heavy metalcoordination compounds which form covalent DNA adducts. Generally, theseheavy metal compounds bind covalently to DNA to form, in pertinent part,cis-1,2-intrastrand dinucleotide adducts. Generally, this class isrepresented by cis-diamminedichloroplatinum (II) (cisplatin), andincludes cis-diammine-(1,1-cyclobutanedicarboxylato) platinum(II)(carboplatin), cis-diammino-(1,2-cyclohexyl) dichloroplatinum(II), andcis-(1,2-ethylenediammine) dichloroplatinum(II). Platinum first agentsinclude analogs or derivatives of any of the foregoing representativecompounds.

[0061] Tumors currently manageable by platinum coordination compoundsinclude testicular, endometrial, cervical, gastric, squamous cell,adrenocortical and small cell lung carcinomas along withmedulloblastomas and neuroblastomas. Trans-Diamminedichloroplatinum (II)(trans-DDP) is clinically useless owing, it is thought, to the rapidrepair of its DNA adducts. The use of trans-DDP as a chemotherapeuticagent herein likely would provide a compound with low toxicity innonselected cells, and high relative toxicity in selected cells. In apreferred embodiment, the platinum compound is cisplatin.

[0062] The invention being thus described, practice of the invention isillustrated by the experimental examples provided below. The skilledpractitioner will realize that the materials and methods used in theillustrative examples can be modified in various ways. Suchmodifications are considered to fall within the scope of the presentinvention.

EXAMPLES Example 1 RNA Isolation from FPE Tissue

[0063] RNA is extracted from paraffin-embedded tissue by the followinggeneral procedure.

[0064] A. Deparaffinization and hydration of sections:

[0065] (1) Aportion of an approximately 10 μM section is placed in a 1.5mL plastic centrifuge tube.

[0066] (2) 600 μL, of xylene are added and the mixture is shakenvigorously for about 10 minutes at room temperature (roughly 20 to 25°C.).

[0067] (3) The sample is centrifuged for about 7 minutes at roomtemperature at the maximum speed of the bench top centrifuge (about10-20,000×g).

[0068] (4) Steps 2 and 3 are repeated until the majority of paraffin hasbeen dissolved. Two or more times are normally required depending on theamount of paraffin included in the original sample portion.

[0069] (5) The xylene solution is removed by vigorously shaking with alower alcohol, preferably with 100% ethanol (about 600 μL) for about 3minutes.

[0070] (6) The tube is centrifuged for about 7 minutes as in step (3).The supernatant is decanted and discarded. The pellet becomes white.

[0071] (7) Steps 5 and 6 are repeated with successively more diluteethanol solutions: first with about 95% ethanol, then with about 80% andfinally with about 70% ethanol.

[0072] (8) The sample is centrifuged for 7 minutes at room temperatureas in step (3). The supernatant is discarded and the pellet is allowedto dry at room temperature for about 5 minutes.

[0073] B. RNA Isolation with Phenol-Chloroform

[0074] (1) 400 μL guanidine isothiocyanate solution including 0.5%sarcosine and 8 μL dithiothreitol is added.

[0075] (2) The sample is then homogenized with a tissue homogenizer(Ultra-Turrax, IKA-Works, Inc., Wilmington, N.C.) for about 2 to 3minutes while gradually increasing the speed from low speed (speed 1) tohigh speed (speed 5).

[0076] (3) The sample is then heated at about 95° C. for about 5-20minutes. It is preferable to pierce the cap of the tube containing thesample with a fine gauge needle before heating to 95 ° C. Alternatively,the cap may be affixed with a plastic clamp or with laboratory film.

[0077] (4) The sample is then extracted with 50 μL 2M sodium acetate atpH 4.0 and 600 μL of phenol/chloroform/isoamyl alcohol (10:1.93:0.036),prepared fresh by mixing 18 mL phenol with 3.6 mL of a 1:49 isoamylalcohol:chloroform solution. The solution is shaken vigorously for about10 seconds then cooled on ice for about 15 minutes.

[0078] (5) The solution is centrifuged for about 7 minutes at maximumspeed. The upper (aqueous) phase is transferred to a new tube.

[0079] (6) The RNA is precipitated with about 10 μL glycogen and with400 μL isopropanol for 30 minutes at −20° C.

[0080] (7) The RNA is pelleted by centrifugation for about 7 minutes ina benchtop centrifuge at maximum speed; the supernatant is decanted anddiscarded; and the pellet washed with approximately 500 μL of about 70to 75% ethanol.

[0081] (8) The sample is centrifuged again for 7 minutes at maximumspeed. The supernatant is decanted and the pellet air dried. The pelletis then dissolved in an appropriate buffer for further experiments(e.g., 50 μl. 5 mM Tris chloride, pH 8.0).

Example 2 mRNA Reverse Transcription and PCR

[0082] Reverse Transcription: RNA was isolated from microdissected ornon-microdissected formalin fixed paraffin embedded (FPE) tissue asillustrated in Example 1 and as previously described in U.S. applicationSer. No. 09/469,338 filed Dec. 20, 1999, which is hereby incorporated byreference in its entirety. After precipitation with ethanol andcentrifugation, the RNA pellet was dissolved in 50 ul of 5 mM Tris/Cl atpH 8.0. M-MLV Reverse Transcriptase will extend an oligonucleotideprimer hybridized to a single-stranded RNA or DNA template in thepresence of deoxynucleotides, producing a complementary strand. Theresulting RNA was reverse transcribed with random hexamers and M-MLVReverse Transcriptase from Life Technologies. The reverse transcriptionwas accomplished by mixing 25 μl of the RNA solution with 25.5 μl of“reverse transcription mix” (see below). The reaction was placed in athermocycler for 8 min at 26° C. (for binding the random hexamers toRNA), 45 min at 42° C. (for the M-MLV reverse transcription enzymaticreaction) and 5 min at 95° C. (for heat inactivation of DNAse).

[0083] “Reverse transcription mix” consists of 10 ul 5×buffer (250 mMTris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.5 ul random hexamers (50O.D. dissolved in 550 ul of 10 mM Tris-HCl pH 7.5) 5 ul 10 mM dNTPs(dATP, dGTP, dCTP and dTTP), 5 ul 0.1 M DTT, 1.25 ul BSA (3mg/ml in 10mM Tris-HCL, pH 7.5), 1.25 ul RNA Guard 24,800 U/ml (RNAse inhibitor)(Porcine #27-0816, Amersham Pharmacia) and 2.5 ul MMLV 200 U/ul (LifeTech Cat #28025-02).

[0084] Final concentrations of reaction components are: 50 mM Tris-HCl,pH 8.3, 75 mM KCl, 3 mM MgCl2, 1.0 mM dNTP, 1.0 mM DTT, 0.00375. mg/mlBSA, 0.62 U/ul RNA Guard and 10 U/ ul MMLV.

[0085] PCR Quantification of mRNA expression. Quantification of GST-picDNA and an internal control or house keeping gene (e.g., β-actin) cDNAwas done using a fluorescence based real-time detection method (ABIPRISM 7700 or 7900 Sequence Detection System [TaqMan®], AppliedBiosystems, Foster City, Calif.) as described by Heid et al., (GenomeRes 1996;6:986-994); Gibson et al., (Genome Res 1996;6:995-1001). Inbrief, this method uses a dual labelled fluorogenic TaqMan®oligonucleotide probe, (GST-219T (SEQ ID NO: 3), T_(m)=69° C.), thatanneals specifically within the forward and reverse primers. Laserstimulation within the capped wells containing the reaction mixturecauses emission of a 3′quencher dye (TAMRA) until the probe is cleavedby the 5′ to 3′nuclease activity of the DNA polymerase during PCRextension, causing release of a 5′ reporter dye (6FAM). Production of anamplicon thus causes emission of a fluorescent signal that is detectedby the TaqMan®'s CCD (charge-coupled device) detection camera, and theamount of signal produced at a threshold cycle within the purelyexponential phase of the PCR reaction reflects the starting copy numberof the sequence of interest. Comparison of the starting copy number ofthe sequence of interest with the starting copy number of theinternalcontrol gene provides a relative gene expression level. TaqMan® analysesyield values that are expressed as ratios between two absolutemeasurements (gene of interest/internal control gene).

[0086] The PCR reaction mixture consisted 0.5 μl of the reversetranscription reaction containing the cDNA prepared as described above600 nM of each oligonucleoride primer (GST-F (SEQ ID NO:1), T_(m)=59° C.and GST-R (SEQ ID NO: 2), T_(m)=59° C.), 200 nM TaqMan® probe (SEQ IDNO:3), 5 U AmpliTaq Gold Polymerase, 200 μM each dATP, dCTP, dGTP, 400μM dTTP, 5.5 mM MgCl₂, and 1×TaqMan® Buffer A containing a referencedye, to a final volume of less than or equal to 25 μl (all reagentsApplied Biosystems, Foster City, Calif.). Cycling conditions were, 95°C. for 10 min, followed by 45 cycles at 95° C. for 15 s and 60° C. for 1min Oligonucleotides used to quantify internal control gene β-Actin wereβ-Actin TaqMan® probe (SEQ ID NO: 4), β-Actin-592F (SEQ ID NO: 5) andβ-Actin-651R (SEQ ID NO: 6).

[0087] The oligonucleotide primers GST-F (SEQ ID NO:1) and GST-R (SEQ IDNO: 2), used in the above described reaction will amplify a 72 bpproduct.

Example 3 Determining the Uncorrected Gene Expression (UGE) for GST-pi

[0088] Two pairs of parallel reactions are carried out, i.e., “test”reactions and the “calibration” reactions. The GST-pi amplificationreaction and the β-actin internal control amplification reaction are thetest reactions. Separate GST-pi and β-actin amplification reactions areperformed on the calibrator RNA template and are referred to as thecalibration reactions. The TaqMan® instrument will yield four differentcycle threshold (Ct) values: Ct_(GST-p) and Ct_(β-actin) from the testreactions and Ct_(GST-pi) and Ct_(β-actin) from the calibrationreactions. The differences in Ct values for the two reactions aredetermined according to the following equation:

ΔCt _(test) =Ct _(GST-pi) −Ct _(β-actin)  (From the “test” reaction)

ΔCt _(calibrator) =Ct _(GST=pi) −Ct _(β-actin)  (From the “calibration”reaction)

[0089] Next the step involves raising the number 2 to the negative ΔCt,according to the following equations.

2^(−ΔCt) ^(_(test))   (From the “test” reaction)

2^(−ΔCt) ^(_(calibrator))   (From the “calibration” reaction)

[0090] In order to then obtain an uncorrected gene expression for GST-pifrom the TaqMan® instrument the following calculation is carried out:

Uncorrected gene expression (UGE) for GST-pi=2^(−ΔCt) ^(_(test))/2^(−ΔCt) ^(_(calibrator))

Normalizing UGE with known relative GST-pi Expression Levels

[0091] The normalization calculation entails a multiplication of the UGEwith a correction factor K_(GST-pi)) specific to GST-pi and a particularcalibrator RNA. A correction factor K_(GST-pi) can also be determinedfor any internal control gene and any accurately pre-quantifiedcalibrator RNA. Preferably, the internal control gene β-actin and theaccurately pre-quantified calibrator Adult Colon, Disease Human TotalRNA, (Cat. No. #735263) from Stratagene, are used. Given these reagentscorrection factor K_(GST-pi) equals 7.28×10⁻³.

[0092] Normalization is accomplished using a modification of the ΔCtmethod described by Applied Biosystems, the TaqMan® manufacturer, inUser Bulletin #2 and described above. To carry out this procedure, theUGE of 6 different test tissues was analyzed for GST-pi expression usingthe TaqMan® methodology described above. The internal control geneβ-actin and the calibrator RNA,Adult Colon, Disease Human Total RNA,(Cat. No. #735263) from Stratagene was used.

[0093] The known relative GST-pi expression level of each sample 14-1,14-5, 14-8, 13-24, 13-25 was divided by its corresponding TaqMan®derived UGE to yield an unaveraged correction factor K.

K_(unaveraged)=Known Values/UGE

[0094] Next, all of the K values are averaged to determine a singleK_(GST-p) correction factor specific for GST-pi, Adult Colon, DiseaseHuman Total RNA, (Cat. No. #735263) from Stratagene from calibrator RNAand β-actin.

[0095] Therefore, to determine the Corrected Relative GST-pi Expressionin an unknown tissue sample on a scale that is consistent withpre-TaqMan® GST-pi expression studies, one merely multiplies theuncorrected gene expression data (UGE) derived from the TaqMan®apparatus with the K_(GST-pi) specific correction factor, given the useof the same internal control gene and calibrator RNA.

Corrected Relative GST-pi Expression=UGE×K_(GST-pi)

[0096] A K_(GST-pi) may be determined using any accuratelypre-quantified calibrator RNA or internal control gene. Future sourcesof accurately pre-quantified RNA can be calibrated to samples with knownrelative GST-pi expression levels as described in the method above ormay now be calibrated against a previously calibrated calibrator RNAsuch as Adult Colon, Disease Human Total RNA, (Cat. No. #735263) fromStratagene described above.

[0097] For example, if a subsequent K_(GST-pi) is determined for adifferent internal control gene and/or a different calibrator RNA, onemust calibrate both the internal control gene and the calibrator RNA totissue samples for which GST-pi expression levels relative to thatparticular internal control gene have already been determined. Such adetermination can be made using standard pre-TaqMan®, quantitativeRT-PCR techniques well known in the art. The known expression levels forthese samples will be divided by their corresponding UGE levels todetermine a K for that sample. K values are then averaged depending onthe number of known samples to determine a new K_(GST-pi) specific tothe different internal control gene and/or calibrator RNA.

Example 4 GST-pi Expression Correlates with Survivability

[0098] Total mRNA was isolated from microdissected FPE pretreatmenttumor samples, and Corrected Relative GST-pi Expression was measuredusing quantitative RT-PCR as described in Examples 2 and 3. A method formRNA isolation from such samples is described in Example 1 and in U.S.patent application Ser. No. 09/469,338, filed Dec. 20, 1999, and ishereby incorporated by reference in its entirety.

[0099] The values of the gene expressions were correlated with clinicaloutcome using appropriate statistical methods. Survival was estimatedaccording to Kaplan and Meier (Kaplan et al., J Am Stat Assoc 1958; 53:187-220). Univariate analysis was performed with the log-rank test(Mantel, Chemother Rep 1966; 50: 163-170). The level of significance wasset to P<0.05. All P values reported were based on two-sided tests.

[0100] A total of 31 esophageal or gastroesophageal junction(esophagocardiac) adenocarcinoma tumor specimens from 31 patients wereanalysed for GST-pi mRNA expression analysis. Thirty (97%) of thepatients were male, the median age was 64 years (mean 60.9 years, range36-78 years). The ethnic background of this group included 29Caucasians, 1 Asian, and 1 African-American. Using TNM clinical stagingcriteria, 2 (6.5%) of the patients had Stage I disease, 22 (71%) hadStage II disease, 1 (3.2%) had Stage III disease, and 6 (19.4%) patientshad Stage IV disease. Overall survival was assessable for all patients.The median overall survival was 17.17 months (mean 24.8 months, range3.8-156.7 months). Twelve (38.7%) of the patients had died and 19(61.3%) were alive.

[0101] The treatment consisted of all patients receiving two cycles of5-FU given as 800 mg/m² per day for 5 days or 1000 mg/m² per day for 4days plus 75 mg/m² cisplatin with concurrent 45 Gy radiation, followedby operative resection. For entry into the study, each patient had tohave completed the chemotherapy regimen and the prescribed radiotherapy,undergone a gross complete resection, and lived at least 30 days aftersurgery.

[0102] The influence of tumor stage was accounted for by pooling thedata over the TNM stage strata. Survival curves and log-rank statisticswere generated for Stage II and Stage IV disease patients only becauseof the very small numbers of patients in the other stages. The mediancorrected relative GST-pi mRNA expression level was 1.0×10⁻³ (mean0.51×10⁻³, range 0.0-16.1×10⁻³, all values GST-pi×10⁻³/β-actin). Ananalysis of survival according to GST-pi values showed that patientswhose tumors had a relative GST-pi gene expression level higher than themedian value had a statistically significant survival benefit comparedto those with levels below the median value (P=0.0073, log-rank test).Accordingly, the median corrected relative GST-pi mRNA was assigned tobe a threshold value. This association is shown graphically in FIG. 1.The relationship was independent of stage. FIGS. 2 and 3 show that theassociation was present if the analysis was confined only to those withStage II or Stage IV disease.

[0103] GST-pi mRNA expression is a significant prognostic factor forpatients with esophagocardiac adenocarcinoma who are treated with acisplatin-containing regimen.

1 6 1 24 DNA Artificial Sequence Primer 1 cctgtaccag tccaatacca tcct 242 20 DNA Artificial Sequence Primer 2 tcctgctggt ccttcccata 20 3 20 DNAArtificial Sequence Probe 3 tcacctgggc cgcacccttg 20 4 18 DNA ArtificialSequence Probe 4 accaccacgg ccgagcgg 18 5 18 DNA Artificial SequenceProbe 5 tgagcgcggc tacagctt 18 6 20 DNA Artificial Sequence Probe 6tccttaatgt cacgcacgat 20

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. A method for determining the level of GST-piexpression in a fixed paraffin embedded tissue sample comprising; (a)deparaffinizing the tissue sample; to obtain a deparaffinized sample;(b) isolating mRNA from the deparaffinized sample in the presence of aneffective amount of a chaotropic agent; (c) subjecting the mRNA toamplification using a pair of oligonucleotide primers that hybridizeunder stringent conditions to a region of the GST-pi gene, to obtain anamplified sample; (d) determining the quantity of GST-pi mRNA relativeto the quantity of an internal control gene's mRNA.
 9. The method ofclaim 8 wherein, the pair of oligonucleotide primers consists of theoligonucleotide primer pair GST or a pair of oligonucleotide primers atleast or about 80% identical thereto.
 10. The method of claim 8 wherein,the internal control gene is β-actin.
 11. The method of claim 8 wherein,mRNA isolation is carried out by (a) heating the tissue sample in asolution comprising an effective concentration of a chaotropic compoundto a temperature in the range of about 75 to about 100° C. for a timeperiod of about 5 to about 120 minutes; and (b) recovering said mRNAfrom said chaotropic solution.
 12. (canceled)
 13. (canceled) 14.(canceled)